Novel Applications of Acridinium Compounds and Derivatives in Homogeneous Assays

ABSTRACT

Chemiluminescent acridinium compounds are used in homogeneous assays to determine the concentration of an analyte in a sample without strong acid or strong base treatment. The chemiluminescent acridinium compounds include acridinium esters with electron donating functional groups at the C2 and/or C7 position on the acridinium nucleus to inhibit pseudo-base formation, or acridinium sulfonamides with or without electron donating functional groups at the C2 and/or C7 position on the acridinium nucleus.

This application is a divisional of application Ser. No. 10/260,504filed Sep. 27, 2002, the contents of which are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the application of acridinium compoundswith certain specific structural features in homogeneous assays.Important structural features that are necessary to ensure lightemission at mild pH are disclosed herein.

2. Background of the Invention

Chemiluminescent acridinium esters (AE) are extremely useful labels thathave been used extensively in immunoassays and nucleic acid assays. U.S.Pat. Nos. 4,745,181; 4,918,192; 5,110,932; 5,241,070; 5,538,901;5,663,074 and 5,656,426 disclose a variety of stable acridinium esterswith different functional groups for conjugation to a variety ofbiologically active molecules referred to as analytes.

U.S. Pat. No. 5,656,426 discloses a hydrophilic acridinium ester withincreased quantum yield. Considerable effort has also been directedtowards the design of acridinium esters whose emission wavelength can bealtered by either incorporating unique structural features in theacridinium ester or by employing the principle of energy transfer. SeeU.S. Pat. Nos. 5,395,752; 5,702,887; 5,879,894; 6,165,800; and6,355,803.

Acridinium sulfonamides are another class of chemiluminescent compoundswhere the substituted phenolic leaving group is replaced with asubstituted sulfonamide. The synthesis and applications of theseacridinium compounds in heterogeneous assays have been described in theprior art: Adamezyk et al, Tetrahedron, vol. 55, pages 10899-10914(1999); Mattingly, J. Biolumin. Chemilumin., vol. 6, pages 107-114(1991); and Adamcyzk et al, Bioconjugate Chem. vol. 11, pages 714-724(2000).

Mechanistically, acid treatment converts the pseudo-base form of theacridinium compound to the acridinium ester which can then participatein the chemiluminescent reaction with hydrogen peroxide. The addition ofalkali serves not only to neutralize the acid but also to raise the pHof the reaction medium for the ionization of hydrogen peroxide.

The relatively harsh reagents with strong acidic pHs on the order ofless than 2 and strong basic pHs on the order of greater than 12 thatare required for triggering chemiluminescence as described above aredetrimental to the preservation of binding complexes such, asantibody-hapten complexes or nucleic acid hybrids. This is not a problemin a heterogeneous assay format where signal generation is typicallyperformed at the end of the assay and unbound tracer and/or interferingsubstances have been removed.

A homogeneous assay is an analytical method where measurement of asubstance of interest is performed without any separation procedures. Ina homogeneous assay format, in order to detect the occurrence of bindingevents using chemiluminescent acridinium compounds as tags, lightgeneration must occur under milder pH conditions because harsh pHconditions are detrimental to the preservation of binding complexes.Additionally, a mechanism to distinguish between bound and unboundtracer or analyte is needed because no separation is performed in ahomogeneous assay. These constraints have hampered the utility ofacridinium compounds in homogeneous assays.

The only homogeneous assays using acridinium compounds that are believedto have been described in the literature are “hybridization protectionassays”. See Nelson et al, Biochemistry vol. 35, pages 8429-8438 (1996).In these assays, the acridinium ester portion of an acridiniumester-labeled nucleic acid probe is selectively decomposed by hydrolysiswhen it is not hybridized to a target. Hybridization of the probe to itstarget results in protection of the acridinium ester from hydrolysisthereby enabling the distinction between hybridized versusnon-hybridized DNA. By employing acridinium esters with similarhydrolysis rates but different time-dependent light emission profiles,homogeneous multi-analyte assays were devised to detect nucleic acids.

In Nelson et al., the structure of the acridinium ester is not beingaltered to enable light emission under mild conditions. Rather, theNelson et al. assay takes advantage of the different rates of hydrolysis(degradation) of the acridinium ester when a nucleic acid labeled withthe acridinium ester is either free or is hybridized to thecomplementary sequence of the acridinium ester-labeled nucleic acid.Thus, the property of the nucleic acid, whether or not it is hybridizedto its complementary sequence, is being used to alter the rate ofdegradation of the acridinium ester.

Fluorescence Resonance Energy Transfer (FRET) is a well-known phenomenonthwart has been widely used to study proximity effects in biomolecules.In FRET, an electronically excited fluorescent donor molecule transfersits electronic energy to a second, acceptor molecule throughdipole-dipole coupling. This energy transfer causes fluorescencequenching of the donor. If the acceptor is fluorescent, its fluorescenceis then observed.

The efficiency of energy transfer is inversely proportional to the sixthpower of the distance separating the donor and acceptor fluors and alsodepends directly on the fluorescent quantum yield of the donor and theextinction coefficient of the acceptor at the wavelength of maximalemission of the donor. Because of the distance dependence, FRET isnormally not observed at distances >10 nm. Homogeneous immunoassaysbased on chemiluminescence energy transfer have also been describedusing isoluminol as the chemiluminescent donor and fluorescein as theacceptor. See Patel et al, Clin. Chem. vol. 2919, pages 1604-1608(1983). Assays for small analytes such as progesterone as well asprotein antigens were constructed using the isoluminol-fluorescein,donor-acceptor pair presumably because chemiluminescence from isoluminolcan be triggered under mild conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 10 are all graphical representations.

FIG. 1 depicts pH titrations of dimethyl acridinium ester (DMAE);

FIG. 2 depicts pH titrations of 2,7-dimethoxy-DMAE;

FIG. 3 depicts a homogeneous carbamazepine assay using an acridiniumester tracer;

FIG. 4 depicts a homogeneous carbamazepine assay using an acridiniumsulfonamide tracer;

FIG. 5 depicts a homogeneous theophylline assay using an acridiniumester tracer;

FIG. 6 depicts a homogeneous theophylline assay using an acridiniumsulfonamide tracer;

FIG. 7 depicts a homogeneous valproate assay using an acridinium estertracer;

FIG. 8 depicts a homogeneous valproate assay using an acridiniumsulfonamide tracer;

FIG. 9 depicts a homogeneous valproate assay using quenching;

FIG. 10 depicts a CL RET from Biotin-AE to Biotin-jfNpFL in astreptavidin complex.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention comprises certain acridinium compounds includingacridinium esters and acridinium sulfonamides whose chemiluminescencecan be triggered under relatively mild pH conditions. The availabilityof such acridinium compounds whose chemiluminescence can be triggeredunder relatively mild conditions makes it possible to use such compoundsin homogeneous resonance energy transfer assays. It has beenunexpectedly discovered, that hapten tracers derived from theseacridinium compounds show significant differences in their efficiency oflight production depending upon whether they are free in solution orbound to their respective antibodies. The discovery of this phenomenonhas enabled the production of homogeneous assays for clinicallyimportant analytes.

Hapten tracers derived from the inventive acridinium compounds uponbinding to their corresponding antibodies exhibit an alteration in theefficiency of light emission. Efficiency of light emission means thatthe acridinium compounds emit more light or less light depending uponwhether they are free or are bound to antibodies. For acridinium esters,light emission is increased whereas for acridinium sulfonamides, lightemission is decreased when these compounds are bound to antibodies.Therefore, depending upon the acridinium compound, light emission iseither enhanced or attenuated.

These effects have been utilized to devise homogeneous assays fordetermining the concentration or quantitation or detection of suchclinically important analytes, including theophylline, valproate andcarbamazepine. In addition, the acridinium compounds disclosed hereincan also be used in resonance energy transfer (RET) assays where excitedstate energy from the acridone formed from the acridinium compoundduring the chemiluminescent reaction is transferred in an intermolecularfashion to an acceptor molecule such as naphthofluorescein and dabsylwhose spectral properties (emission at a different wavelength orquenching) are distinguishable from that of the acridinium compound.

Acridinium Structure and Pseudo-Base Formation

It is well known that acridinium compounds exist in an equilibriumbetween the acridinium form and the pseudo-base form in aqueous mediawhere virtually all immunoassays are performed. The pseudo-base form ofacridinium compounds cannot react with hydrogen peroxide and thus cannotproduce chemiluminescence. The equilibrium between the chemiluminescentacridinium form and non-chemiluminescent pseudo-base form is stronglyinfluenced by the pH of the medium. Acidic pH promotes the formation ofthe acridinium form and basic pH promotes the formation of thepseudo-base form. The following equilibrium reaction shows theconversion of the acridinium compound to the pseudo-base:

Acridinium-Pseudo-Base Equilibrium

R is typically an alkyl, alkenyl, alkynl or aralkyl containing up to 20heteroatoms; or a sulfopropyl or sulfobutyl group; L is typically aleaving group such as an aromatic alcohol or a sulfonamide

In heterogeneous assay formats, chemiluminescence from acridiniumcompounds or acridinium compound-labeled biologically active molecules,referred to as tracers or conjugates, is normally triggered by thesequential addition of two reagents.

An initial treatment of the acridinium compound with a strong acidcontaining peroxide is necessary to convert the pseudo-base form ofacridinium compounds to the acridinium form. The subsequent treatmentwith alkali solution then neutralizes the acid and raises the pH of thereaction medium for the ionization of hydrogen peroxide to allow lightemission to take place.

In a homogeneous assay format, light emission occurs from acridiniumcompounds under mild conditions, that is, at a mild pH of about 6 to 10,preferably about 7 to 9. The maintenance of the acridinium form withoutaddition of a strong acid to the assay medium is a prerequisite togenerate chemiluminescence. Moreover, by omitting the acid-treatmentstep, the need for adding strong base to neutralize the acid and totrigger the chemiluminescence reaction is also obviated.

It has been unexpectedly discovered that acridinium esters bearingelectron donating functional groups at the C-2 and/or C-7 positions onthe acridinium nucleus are less prone to pseudo-base formation thanunsubstituted acridinium esters at the mild pH.

The chemiluminescent acridinium compounds suitable for use in thepresent invention have the following structure:

wherein,

R₁ is an alkyl, alkenyl, alkynyl or aralkyl containing up to 20heteroatoms; or a sulfopropyl or sulfobutyl group;

X is oxygen or nitrogen;

Y is a branched or straight-chained alkyl containing up to 20 carbonatoms, halogenated or unhalogenated, or a substituted aryl, orheterocyclic ring system; when X is oxygen, Z is omitted and Y is apolysubstituted aryl moiety of the formula:

wherein R₄ and R₈ are the same or different, and are hydrogen, alkyl,alkenyl, alkynyl, alkoxyl (—OR), alkylthiol (—SR), or substituted aminogroups that serve to stabilize the —COX— linkage between the acridiniumnucleus and the Y moiety, through steric and/or electronic effect;

R₅ and R₇ are hydrogen or the same as R;

R₆=—R₉—R₁₀,

where R₉ is not required and is branched or straight-chained alkyl,substituted or unsubstituted aryl or aralkyl containing up to 20heteroatoms, and

R₁₀ is a leaving group or an electrophilic functional group attachedwith a leaving group selected from the group consisting of:

a halide or —COOH;

R₅ and R₆, and R₆ and R₇ are interchangeable;

when X is nitrogen, Z is —SO₂—Y′, and Y′ has the same definition of Yand both can be the same or different;

W1 and W2 are the same or different and are electron-donating groups,comprising OR, OH, SR, SH, NH₂, NR′R″; wherein R, R′ and R″ can be thesame or different, and are selected from the group consisting of alkyl,alkenyl, alkynyl, aryl, and aralkyl containing up to 20 heteroatoms;

A⁻ is a counter ion which is introduced to pair with the quaternarynitrogen of said acridinium nucleus, and is selected from the groupconsisting of CH₃SO₄ ⁻, FSO₃ ⁻, CF₃SO₄ ⁻, C₄F₉SO₄ ⁻, CH₃C₆H₄SO₃ ⁻,halide, CF₃COO⁻, CH₃COO⁻, and NO₃ ⁻.

More specifically, the acridinium compound can be an acridinium ester ofthe following structure:

wherein R11 is selected from the following:

—NH—R—NH2, OH, and R1, R and A− are as already described.

Alternatively, the acridinium compound can be an acridinium ester of thefollowing structure:

wherein R₁₂ is selected from hydrogen, amino, hydroxyl, halide, nitro,—CN, —SO₃H, —SCN, —OR, NHCOR, —COR, —COOH, —COOR, or —CONHR; and R₁, A⁻and R₁₁ are as already described.

The acridinium compound of the present invention can also be anacridinium sulfonamide of the following structure:

wherein R₁, A−, R and R₆ are as already described.

In FIG. 1, pH titrations of dimethyl acridinium ester (DMAE) and in FIG.2, pH titrations of 2,7-dimethoxy-DMAE are shown where the UV-absorptionband of the acridinium chrompohore is plotted as a function of pH. Thedecrease in intensity of this absorption band is indicative of thedisruption of the acridinium chromophore and the formation ofpseudo-base. It is evident from FIG. 1 for DMAE that pseudo-baseformation is essentially complete at pH >4. It is evident from FIG. 2that for the electron-rich, 2,7-dimethoxy analog, pseudo-base formationis complete only at pH >11.

In order to determine whether chemiluminescence from electron-richacridinium compounds can be triggered without acid pre-treatment and atmilder pH, light emission from several acridinium compounds including2,7-dimethoxy-DMA and 2-CME-7-methoxy-DMAE and anti-TSH proteinconjugates were measured by employing a single chemical reagentcomprising 1% hydrogen peroxide and 0.5% cetyltrimethylammonium chloride(CTAC) surfactant in sodium carbonate buffer either at pH 8.3 or at pH9.

The results are summarized in Table 1. For all the compounds listed, theamount of light emitted using the modified triggers is expressed as apercentage of the light that is normally measured following thesequential addition of 0.5% hydrogen peroxide in 0.1 N nitric acidfollowed by 0.25 N NaOH containing the CTAC surfactant. Also included inthe table are results for an acridinium sulfonamide. Acridiniumsulfonamides are comparable to acridinium esters in their quantumefficiency of light production and hydrolytic stability.

TABLE 1 Chemiluminescence from Acridinium Compounds % SignalChemiluminescence Compound Retained Triggering Reagent DMAE 0.14 1%H₂O₂ + 0.5% CTAC in 0.2 M sodium carbonate, pH 9 2,7-dimethoxy- 9.5 1%H₂O₂ + 0.5% CTAC in DMAE 0.2 M sodium carbonate, pH 9 2-CME-7-methoxy-43 1% H₂O₂ + 0.5% CTAC in DMAE 0.2 M sodium carbonate, pH 8.3 NSP-AS 6.31% H₂O₂ + 0.5% CTAC in 0.2 M sodium carbonate, pH 8.3 NSP-AS 13.4 1%H₂O₂ + 0.5% CTAC in 0.2 M sodium carbonate, pH 9 2-hydroxy-DMAE 5.8 1%H₂O₂ + 0.5% CTAC in 0.2 M sodium carbonate, pH 9 DMAE-anti-TSH 0.18 1%H₂O₂ + 0.5% CTAC in 0.2 M sodium carbonate, pH 9 2,7-dimethoxy-DMAE- 601% H₂O₂ + 0.5% CTAC in anti-TSH 0.2 M sodium carbonate, pH 9Chemiluminescence was measured for 5 seconds on a Magic Lite AnalyzerLuminometer (MLA1, Bayer Diagnostics). Samples of the various compoundswere prepared in 10 nm phosphate pH 8 containing 150 mM NaCl, 0.05% BSAand 0.01% sodium azide.

The data in Table 1 indicates that the placement of electron donatingfunctional groups at the C-2 and/or C-7 positions enables thechemiluminescent reaction from the corresponding acridinium ester tooccur at a mild pH range of about 8 to 9. The C2 and C7 positions on theacridinium molecule are equivalent. Thus, where there is only onefunctional group, for example, methoxy or hydroxy, at either of thesepositions, it would be referred to as being in the C2 position. If thereare two functional groups, then the second corresponding position is C7.Because the acridinium molecule is symmetrical, the C2 position is thesame as C7. Similarly the C3 position is the same as C6. However, C2 andC3 are not equivalent. Thus a single methoxy group at C2 is notequivalent to a methoxy group at C3.

The chemical structure of the acridinium compounds listed in Table 1 areas follows:

Thus, 2-hydroxy-DMAE and 2,7-dimethoxy-DMAE are each acridinium esterswith electron-donating groups. The hydroxy group in 2-hydroxy-DMAE isthe electron-donating group whereas the two methoxy groups in2,7-dimethoxy-DMAE are electron-donating groups. In addition, even inthe absence of an electron-rich acridinium nucleus, chemiluminescencefrom the acridinium sulfonamide can be triggered at mild pH. It is notknown why this is possible for the acridinium sulfonamide (NSP-AS) evenin the absence of electron-donating groups.

Homogeneous Assays Using Signal Modulation of Antibody-Bound Tracer

(a) Acridinium Esters

The acridinium ester, 2,7-dimethoxy-DMAE was conjugated to threeanalytes: valproate, theophylline and carbamazepine to form tracers thatare commonly measured by immunochemical techniques. The process stepsfor making conjugates of acridinium compounds to various other moleculesof interest are well known to those skilled in the art of organicsynthesis. A conjugate of an acridinium compound is comprised of theacridinium compound and the molecule of interest with a covalent bondlinking the two moieties. The molecule of interest can be a smallmolecule such as a steroid, a therapeutic drug, a vitamin, a hormone andsmall peptides, or a macromolecule such as a protein, nucleic acid,oligosaccharide, antibody, antibody fragment, cell, virus and syntheticpolymer. Examples of proteins include avidin, strepavidin, neutravidin,receptors, and allergens.

The covalent bond can be an amide bond, an ester bond, an ether bond, abond linking two carbon atoms, and the like. Formation of the covalentbond between the acridinium compound and the molecule of interest isgenerally accomplished by a chemical reaction between complementaryreactive functional groups on the two moieties. For example, an amidebond is made from a carboxylic acid functional group on one molecule andan amino group on the second molecule. Typically, the process involvesconverting the carboxylic acid to a reactive form such as an activeester or an anhydride followed by reaction with the amino group.

It was initially envisioned to label the antibodies corresponding tothese analytes with fluors and perform homogeneous assays usingresonance energy transfer from the acridinium tracer to the fluor on theantibody. However, experimentation revealed that antibody binding to thecorresponding tracer for all three analytes resulted in an unexpectedenhancement in light production for the acridinium ester when measuredover a period of a few seconds. Therefore, it was not necessary to labelthe antibodies corresponding to these analytes with fluors.

The magnitude of the light enhancement for the acridinium ester wasdifferent for the three analytes. This can reflect differences in theaffinities and/or microenvironment of these three different antibodiesfor their respective haptens.

Faster kinetics of light emission from the acridinium esterantibody-bound tracer as compared to the free tracer is one possiblemechanism for the observed increase in chemiluminescent signal.Enhancement of chemiluminescence of an isoluminol-biotin conjugate uponbinding to avidin has been reported by Schroeder et al, Anal. Chem. vol.48, pages 1933-1937 (1976).

For all three analytes, valproate, theophylline and carbamazepine, thechemiluminescent signal from the binding reactions could be measureddirectly without dilution. These small molecule assays are thusextremely simple to perform. The steps involved in devising and carryingout a homogeneous assay for the measurement of a small molecule analyteinclude forming a conjugate of the small molecule analyte and theacridinium compound as already described and making a solution of theconjugate in an appropriate solvent, such as a mixture of an organicsolvent, for example, dimethyl formamide and water or a buffer. Theacridinium compound is either an acridinium ester with electron-donatingfunctional groups at the C2 and/or C7 position or an acridiniumsulfonamide with or without electron-donating functional groups at theC2 and/or C7 position.

The conjugate is used to screen various antibodies of the analyte toselect an antibody which upon binding to the acridinium conjugate causesan alteration in the intensity of light emission from the acridiniumconjugate when its chemiluminescence is triggered at a pH of about 6-10.

The conjugate is used to generate a dose-response curve using a seriesof ‘standards’ which comprise different known concentrations of theanalyte in a fluid, typically serum. Generally this is accomplished bymixing a fixed concentration of the acridinium conjugate to eachstandard in a fixed volume of buffer and adding a limited quantity ofthe desired antibody which is capable of forming a complex with eitherthe analyte or its acridinium conjugate in solution.

The solution containing the analyte, the conjugate and the antibody isthen incubated for a fixed length of time, typically about 10 to 60minutes at room temperature, to form a reaction mixture.

The chemiluminescence of the reaction mixture is then or triggered at apH of about 6 to 10 by adding chemiluminescence triggering reagents suchas a solution of hydrogen peroxide in a buffer such as carbonate,borate, phosphate, and tris. The chemiluminescence is then measured witha luminometer. A dose response curve is then prepared by plotting theobserved chemiluminescence versus the concentration of the analyte inthe standard.

The concentration of an unknown sample of the analyte is then determinedby incubating the sample at the same conditions as the standardsolution. The chemiluminescence of the unknown sample of analyte is thentriggered with the same triggering agent used to trigger thechemiluminescence of the standard analyte and its concentration isdetermined from the dose response curve.

Homogeneous assays for carbamazepine, theophylline and valporate wereperformed using the 2,7-dimethoxy-DMAE tracers in a small volume (0.2mL) of buffer. The tracer at nanomolar concentrations was mixed with thehapten standards in human serum. The binding reactions were initiated bythe addition of a limited quantity of the corresponding antibody. Theseare true competitive assays because the analyte and the tracer competefor a limited amount of antibody.

As the concentration of the hapten increased, the concentration ofantibody-bound tracer decreased and consequently, the chemiluminescentsignal for the acridinium ester was depressed proportionally.

(b) Acridinium Sulfonamides

Chemiluminescent triggering under mild conditions of hapten conjugatesof acridinium sulfonamides showed characteristics that contrastedsharply to what was observed with acridinium esters. For acridiniumesters, tracer binding to the antibody leads to an increase inchemiluminescence whereas for acridinium sulfonamides, tracer binding bythe antibody leads to a decrease in chemiluminescence. Slower kineticsof light emission from the acridinium sulfonamide antibody-bound traceris a possible mechanism for the observed decrease in chemiluminescentsignal. The relative luminescent yield of the acridinium sulfonamidederived tracer was dependent upon the conjugated hapten. In addition,electron donating functional groups are not needed at the C2 and/or C7positions to trigger chemiluminescence.

In comparison to the free tracers, antibody-bound tracers exhibited adecrease in luminescence when their chemiluminescence was measured for afew seconds. The antibody bound valproate, theophylline andcarbamazepine tracers exhibited 28%, 12% and 5% of the relativeluminescence of the free tracers. A kinetic profile of the lightproduction revealed a decrease in the chemiluminescent reaction rateupon antibody binding. This phenomenon was used to devise homogeneousassays for the analytes: valproate, theophylline and carbamazepine.

Homogeneous Assays Using Quenching

In addition to signal modulation due to tracer binding by an antibody,an alternate construct for a homogeneous assay using the acridiniumcompounds of this invention entails using the phenomenon of signalquenching as a mechanism to generate a dose response curve.

Signal quenching reduces the chemiluminescence of the acridiniumcompound by using an acceptor molecule which absorbs energy from theacridinium compound but does not have an emission of its own. Theacridone is the chemical species that is formed in anelectronically-excited state from the chemical reaction of theacridinium compound with hydrogen peroxide. Any molecule that can absorbthe electronic excitation energy of the acridone, which normally isemitted as light, and dissipate it via non-radiative pathways canperform this quenching function. Dabsyl is commonly used for thispurpose.

In the antibody-tracer complex, the spatial proximity of the antibodyand the acridinium compound-derived tracer suggests that excited-stateenergy from the acridinium compound may be transferred to suitableacceptor molecules on the antibody. To investigate this matter further,a valproate tracer of 2-CME-7-methoxy-DMAE was prepared. See Example 2.

This tracer when incubated with an unlabeled valproate antibody did notshow any modulation of its chemiluminescent signal. When the antibodywas labeled with the quencher dabsyl, antibody-bound tracer showed adrop in luminescence. Thus, tracer binding to the dabsyl-labeledantibody could be measured by recording a concomitant decrease inchemiluminescent signal.

In an assay that uses the principle of signal-quenching, all steps arethe same as the homogeneous assay using signal modulation except thatthe conjugate of an antibody and a quencher is used.

A valproate assay was devised using the quenching phenomenon. Thevalproate antibody used previously with the 2,7-dimethoxy-DMAE-valproateconjugate was labeled with the synthetic dabsyl derivativedabsyl-ED-glutarate-NHS ester. The dabsyl derivative was synthesized byfirst condensing dabsyl chloride with ethylene diamine followed byreaction with glutaric anhydride and subsequent conversion of theresulting carboxylic acid to the N-hydroxy succinimide (NHS) ester.

A homogeneous assay using the labeled antibody was performed by allowingthe tracer and the valproate standard in sheep serum to compete for alimited amount of the dabsyl-labeled antibody. An increase in theconcentration of valproate led to an increase in chemiluminescentsignal.

Homogeneous Assays Using Chemiluminescence Resonance Energy Transfer

The chemiluminescent Resonance Energy Transfer (RET) immunoassay isbased on formation of a complex consisting of the analyte with twobinding partners, for example antibodies. Fabs, lectins enzymes andnucleic acid binding ligands can also be employed in an analogousfashion, where one is labeled with the chemiluminescent donor compoundand the second, directed against another epitope on the analyte, islabeled with a fluorophore acceptor. Upon binding, the analyte becomesthe bridge between the donor and the acceptor permitting efficient RETif the two are within less than 10 nm r distance. Triggering thechemiluminescence with the light releasing reagents produces light thatis transferred to the acceptor and measured at its optimum wavelength.

In principle, the analytes measured in the chemiluminescent ResonanceEnergy Transfer immunoassay are macromolecules having at least twobinding sites in the same molecule. Such macromolecules include thegroup consisting of proteins, nucleic acids, oligosaccharides,antibodies, antibody fragments, cells, viruses and synthetic polymers.

The process steps for a chemiluminescent homogeneous resonance energytransfer assay for the measurement of a macromolecular analyte such as aprotein include making a first conjugate of an antibody to the analyteusing the acridinium compounds of the present invention wherein covalentbond formation is carried out between the antibody and functional groupslocated on the acridinium nucleus of the acridinium compound. Theacridinium compound is either an acridinium ester with electron-donatinggroups at the C2 and/or C7 position, or an acridinium sulfonamide withor without electron-donating groups at the C2 and/or C7 position.

A second conjugate of a second antibody to the analyte is made using an“acceptor” molecule which can absorb electronic excitation energy fromthe first acridinium-antibody conjugate. This second antibody must bindto a different region of the analyte but must also bind sufficientlyclose to the first antibody so that energy transfer from the acridiniumcompound and the acceptor can occur. The distance between regions isgenerally ≦10 nm. The acceptor molecule can either re-emit the absorbedenergy from the acridinium compound at a different wavelength of lightor dissipate it as heat.

The first and second conjugates are used to generate a dose-responsecurve using a series of ‘standards’ which comprise different knownconcentrations of the analyte in a fluid, typically serum. This isgenerally accomplished by mixing fixed concentrations of the acridiniumconjugates to each standard in a fixed volume of buffer. The solutioncontaining the analyte, and the two conjugates is called the “reactionmixture” and is incubated for about 10 to 60 minutes at ambient or roomtemperature.

The chemiluminescence of the reaction mixture is triggered in the pHrange of about 6 to 10 by adding chemiluminescence triggering reagentssuch as a solution of hydrogen peroxide in a buffer and measuring thechemiluminescence using a luminometer. The luminometer must be capableof distinguishing chemiluminescent signals emitted at two differentwavelengths. A dose response came is then prepared by plotting theobserved chemiluminescence versus the concentration of the analyte inthe standard.

The concentration of an unknown sample of the analyte is determined byincubating the reaction mixture of the unknown analyte at the sameconditions as the standard analyte reaction mixture. Thechemiluminescence of the unknown reaction mixture is then triggeredusing the same triggering agent and the same conditions used to pre-parethe standard dose response curve. The concentration of the unknownsample is then determined from the dose response curve.

In the resonance energy transfer assay for the analyte HCG described inExample 13, one antibody is conjugated with an acridinium ester whilethe second antibody is conjugated with the fluorescent compoundnaphthofluorescein.

Labeled Biotins: Avidin Complex

A homogeneous resonance energy transfer assay for the measurement of thevitamin biotin can be performed by making a conjugate of biotin to theacridinium compounds of the present invention wherein covalent bondformation is carried out between biotin and functional groups located onthe acridinium nucleus of the acridinium compound. The acridiniumcompound is either an acridinium ester with electron-donating groups; atthe C2 and/or C7 position or an acridinium sulfonamide with or withoutelectron-donating groups at the C2 and/or C7 position.

A second conjugate of biotin is made using a molecule called an“acceptor” which can absorb electronic excitation energy from theacridinium compound-biotin conjugate. The acceptor molecule can eitherre-emit the absorbed energy at a different wavelength of light ordissipate it as heat.

The above conjugates are used to generate a dose-response curve forbiotin using a series of biotin standards which comprise different knownconcentrations of biotin in a fluid, typically serum. Generally this isaccomplished by mixing fixed concentrations of the two biotin conjugatesand neutravidin or streptavidin to each biotin standard in a fixedvolume of buffer. The solution containing biotin, the two conjugates andthe neutravidin or streptavidin, termed the “reaction mixture” isincubated for about 10 to 60 ml minutes at ambient temperature.

The chemiluminescence of the reaction mixture is triggered in the pHrange of about 6 to 10 by adding chemiluminescence triggering reagentssuch as a solution of hydrogen peroxide in buffer and measuring thechemiluminescence using a luminometer. The luminometer must be capableof distinguishing chemiluminescent signals emitted at two differentwavelengths. A dose response curve for biotin is prepared by plottingthe observed chemiluminescence versus the concentration of biotin in thestandard.

The concentration of an unknown sample of biotin is measured by firstincubating the reaction mixture of the unknown sample at the sameconditions used for the standard reaction mixture. The chemiluminescenceof the unknown sample of biotin is then triggered with the sametriggering agents and at the same conditions as the known reactionmixture. The concentration of the unknown sample of biotin is determinedfrom the concentration on the dose response curve corresponding to themeasured chemiluminescence.

A convenient model to test the limits of RET with the acridiniumcompounds of the present invention serving as the donor, and afluorescent acceptor was to attach them to a biotin, a low molecularweight molecule, to form a conjugate. This then allows the resultingconjugates to bind to avidin, a protein with multiple binding sites forbiotin. Because the dimensions of avidins are about 30×40×50 A°, thebound labeled biotin species would be in close proximity therebypermitting RET. Hence, biotin-hexaethyleneglycol-2-CME-7-methoxy-DMAE(biotin-AE) and biotin-jeffamine-napthofluorescein (biotin-NPFL) weresynthesized. CME is carboxymethylether. See Example 10. Example 11details the resonance energy transfer assay for the measurement ofbiotin. In the resonance energy transfer assay for biotin described inExample 12, biotin conjugates of the acridinium ester,2-CME-7-methoxy-DMAE and the fluorescent compound naphthofluorescein areused in conjunction with neutravidin.

Sandwich Assay for HCG

This is a homogeneous assay for HCG using the principle of resonanceenergy transfer. In this assay, an anti-HCG antibody labeled with thenovel acridinium compounds of the present invention, and a secondanti-HCG antibody, directed against another site, labeled with afluorophore form a complex with HCG, a large molecular weight analyte.Several anti-HCG antibodies were labeled to varying extent with2-CME-7-methoxy-DMAE or naphthofluorescein. An anti-HCG- wholo and ananti-HCG-beta pair were bound sufficiently close to each other toproduce RET. As chemiluminescence (480 nm) was triggered, the emissionshifted to the fluorophore maximum measured at >650 nm. A maximum of˜3-fold increase in signal over background was observed at 5000 mIU/mLHCG. The data appear in Table 2.

TABLE 2 Homogeneous CL RET For HCG HCG mIU/mL NET RLU 0 89,445 10092,920 500 114,530 1,000 130,325 2,500 183,180 5,000 242,860 7,500243,025 10,000 232,905 20,000 199,640

In summary, the chemiluminescence of certain acridinium compounds can betriggered at mild pH. Hapten tracers derived from these acridiniumcompounds when complexed to their respective antibodies show either anenhancement (acridinium esters) or attenuation (acridinium sulfonamides)of light emission. This novel discovery enabled the development ofsimple homogeneous assays for such clinically important analytes astheophylline, valproate and carbamazepine. In addition, the acridiniumcompounds described herein can be used in resonance energy transferassays.

Example 1

This example describes the syntheses of the electron rich acridiniumester, 2,7-dimethoxy-DMAE and its hapten conjugates. Therein,2,7-dimethoxy acridine-9-carboxylic acid was synthesized using theprocedure described in Zomer at al., “Synthesis, Chemiluminescence, andStability of Acridinium Ester Labeled Compounds”, Pract. Spectroc.(Lumin. Tech. Biochem. Anal.), vol. 12, pages 505-511 (1991), andcondensed with 4-carboxybenzyl-2,6-dimethylphenol. The resultingacridine ester vas N-methylated with methyl triflate. Removal of thebenzyl ester protecting group exposed the carboxylic acid. Thesubsequent formation of the valproate, carbamazepine and theophyllineconjugates was accomplished via the active NHS ester.

(a) Synthesis of 2,7-dimethoxy-2′,6′-dimethyl-4′-benzyloxycarbonylphenylacridine-9-carboxylate

2,7-Dimethoxyacridine-9-carboxylic acid (0.5 g, 0.177 mmol) in anhydrouspyridine (25 mL) was cooled in an ice bath under a nitrogen atmosphereand treated with p-toluenesulfonyl chloride (0.674 g, 2 equivalents) andafter 10 minutes, 4-carboxybenzyl-2,6-dimethylphenol (0.453 g, 1equivalent) was added. The reaction was warmed to room temperature.After 1-2 hours, am additional 2 equivalents p-toluenesulfonyl chloridewas added along with 0.5 equivalent phenol and pyridine (10-15 mL). Thereaction was stirred at room temperature under a nitrogen atmosphere for48 hours. The solvent was then removed under reduced pressure and theresidue was dissolved in chloroform (50 mL). This solution was washedwith 2% aqueous sodium bicarbonate followed by 2% aqueous ammoniumchloride. The chloroform extract was then dried over magnesium sulfateand evaporated to dryness. The product was purified by TLC on silicausing 5% ethyl acetate, 95% chloroform. Rf=0.5. Yield=0.663 g (72%)

(b) Synthesis of2,7-dimethoxy-2′,6′-dimethyl-4′-benzyloxycarbonylphenyl-10-methylacridinium-9-carboxylate

The acridine ester from (a) above (0.15 g, 0.29 mmol) in dichloromethane(5 ml) was treated with solid sodium bicarbonate (60 mg, 0.71 mmol) andmethyl trifluoromethane sulfonate (0.2 mL, 0.82 mmol). The reaction wasstirred at room temperature for 16 hours and then methanol (5 mL) wasadded and the reaction was filtered through glass wool. HPLC analysis ofthe filtrate on a C18 4.6 mm×30 cm column and using a 30-minute gradientof 10%->100% MeCN/water each containing 0.05% trifluoroacetic acid at aflow rate of 1 mL/min and UV detection at 260 nm, indicated >90%conversion to product eluting at 21 minutes. The filtrate was evaporatedto dryness and the crude product was used as such for the next reaction.

(c) Synthesis of2,7-dimethoxy-2′,6′-dimethyl-4′-carboxyphenyl-10-methylacridinium-9-carboxylate(2,7-dimethoxy-DMAE)

The acridinium ester from (b) above (25 mg) was treated with 30%HBr/AcOH (3 mL) and the reaction was stirred at room temperature for 4hours. The product was precipitated by the addition of ether (˜50 mL).The ether was decanted and the residue was rinsed several times withether and dried by rotary evaporation. Yield=16 mg. HPLC analysis usingthe gradient described above showed product eluting at 15 minutes.

(d) Synthesis of2,7-dimethoxy-2′,6′-dimethyl-4′-[N-succinimidyl]oxycarbonyl-phenyl10-methylacridinium-9-carboxylate (2,7-dimethoxy-DMAE-NHS)

The acridinium carboxylic acid from (c) above (16 mg, 0.03 mmol, bromidecounter ion) was dissolved in 25% anhydrous MeCN, 75% anhydrousdimethylformamide (DMF) (4 mL) and treated with N-hydroxysuccinimide (17mg, 5 equivalents) and dicyclohexyl carbodiimide (DCC) (31 mg, 5equivalents). The reaction was stirred at room temperature under anitrogen atmosphere for 16 hours. HPLC analysis using the gradientdescribed above indicated complete conversion with the product elutingat ˜16 minutes. The product was purified by preparative HPLC using a 20mm×30 cm column. The HPLC fractions containing the product werelyophilized to dryness to afford a bright yellow powder. Yield=12.6 mg.

(e) Synthesis of 2,7-dimethoxy-DMAE-valproate

2,7-Dimethoxy-DMAE-NHS (1 mg, 1.84 umoles) in DMF (0.1 mL) was cooled inan ice bath and treated with 6-amino-2-propyl-hexanoic acid [0.64 mg, 2equivalents, Sidki et al, J. Clin. Chem. Biochem. vol. 26 (2), page 69,(1988)] dissolved in 100 mM sodium carbonate pH 9 (0.1 mL). The reactionwas stirred at room temperature. After 1 hour, HPLC analysis using thegradient described above, indicated complete disappearance of startingmaterial and the formation of a product eluting a few minutes later.This product was purified by preparative HPLC using a 7.8 mm×30 cmcolumn. The HPLC fractions containing product were lyophilized todryness to afford a yellow powder. Yield=1 mg; MALDI-TOF MS 603.1 obs.(601.7 calc.).

(f) Synthesis of 2,7-dimethoxy-DMAE-HD

2,7-Dimethoxy-DMAE (7 mg, 13.3 umoles) was dissolved in a mixture ofanhydrous MeCN/DMF (2 mL, 1:3) and treated with N-hydroxysuccinimide (9mg, 5 equivalents) and DCC (16 mg, 5 equivalents). The reaction wasstirred at room temperature after 3 hours, 5 equivalents each ofN-hydroxysuccinimide and DCC were added. The resulting reaction wasstirred at room temperature for 16 hours. The reaction mixture was thenfiltered through glass wool and treated with 1,6-hexanediamine (HD, 18.3mg, 10 equivalents) dissolved in 100 mM sodium carbonate pH 9 (2 mL).The reaction was stirred at room temperature. After 2 hours, HPLCanalysis using a 4.6×30 cm C18 column and a 40-minute gradient of10%->60% MeCN/water (each with 0.05% TFA) at a flow rate of 1 mL/min andUV detection at 260 nm indicated product eluting at 23 minutes. This waspurified by preparative HPLC using a 20 mm×30 cm column. The HPLCfractions containing product were lyophilized to dryness.

Yield=9.6 mg (94%); MALDI-TOF MS 545.8 obs. (544.7 calc.).

(g) Synthesis of 2,7-dimethoxy-DMAE-HD-theophylline

8-Carboxypropyltheophylline (5 mg, 18.8 umoles, Sigma) was dissolved inanhydrous DMF (1 mL) and treated with N-hydroxysuccinimide (11 mg, 5equivalents) and DCC (20 mg, 5 equivalents). The reaction was stirred atroom temperature for 16 hours. 2,7-Dimethoxy-DMA HD (3.3 mg, 4.3 umoles)was then added as a solution in methanol (0.2 mL) followed byN,N-diisopropylethylamine (3.2 uL, 18.4 umoles). After three hours HPLCanalysis using the 10%->60% gradient described above indicated producteluting at 28 minutes. This product was isolated by preparative HPLCusing a 20 mm×30 cm column. The HPLC fractions containing product werelyophilized to dryness. Yield=2.7 mg (76%); MALDI-TOF MS 794.2 obs.(792.9 calc.).

(h) Synthesis of 2,7-dimethoxy-DMAE-HD-SA-ED-Carbamazepine

Carbamazepine-ED-SA was prepared in two steps from carbamazepine-N-acidchloride that fleas provided by the Central Research Labs of Ciba-GeigyLimited, now Novartis Inc. A solution of carbamazepine-N-acid chloride(2 g, 7.828 mmol) in 10 ml of tetrahydrofuran was dropwise added to asolution of ethylene diamine (5.24 ml, 10 equivalents) in 100 ml ofether at 0° C. The reaction was allowed to stir at room temperature for1.5 hr, and then was evaporated under reduced pressure with the help ofxylene. The solid obtained was transferred to a fritted funnel andwashed with chloroform three times to give carbamazepine-ED as anoff-white material in 955 mg. The carbamazepine-ED (950 mg, 3.42 mmol)was suspended in a mixed solvent of 70 ml of DMF/chloroform (1:1),followed by addition of succinic anhydride (513 mg, 1.5 equivalents) andtriethylamine (1.9 ml, 4 equivalents). The mixture was heated withstirring at 100° C. for an hour to give a homogeneous solution. It wasevaporated under reduced pressure to dryness. The residue was treatedwith 60 ml of water. The suspension was stirred at room temperature 50minutes to form a white precipitate. It was collected and washed withwater and then chloroform.

After it dried under vacuum, 552 mg of carbamazepine-ED-SA was obtained.Carbamazepine-ED-SA (5 mg, 13.2 umoles) in anhydrous DMF (0.5 mL) wastreated with N-hydroxysuccinimide (7.6 mg, 5 equivalents) and DCC (13.6mg, 5 equivalents). The reaction was stirred at room temperature for 3-4hours and then 1.7 mg of the active ester was withdrawn and diluted to900 uL DMF. This solution was treated with a solution of2,7-dimethoxy-DMAE-HD (2.5 mg, 3.24 umoles) in methanol (0.2 mL) alongwith N,N-diisopropylethylamine (2.4 uL, 13.8 umoles). The reaction wasstirred at room temperature for 16 hours. HPLC analysis using the10%->60% gradient described above indicated product eluting at ˜34minutes. The product was purified by preparative HPLC using a 20 mm×30cm column and the HPLC fractions containing product were lyophilized todryness. Yield=1.6 mg (54%); MALDI-TOF MS 907.8 obs. (906.1 calc.).

(i) Synthesis of 2,7-dimethoxy-DMAE-anti-TSH conjugate

Anti-TSH monoclonal antibody (0.5 mg, 3.33 nmoles) in a mixture of 100mM sodium phosphate pH 8 (0.1 mL) and PBS pH 8 (80 uL) was treated witha solution of 2,7-dimethoxy-DMAE-NHS (36.2 ug, 20 equivalents) in DMF(20 uL). The reaction was stirred at 2-4° C. in a cold-box for 16 hoursand then the labeled protein was isolated by Sephadex G25 gel-filtrationchromatography using water as eluent. The conjugate eluting in the voidvolume of the column was collected and concentrated to 1 mL. Thissolution was diluted with 4 mL PBS pH 7.4 also containing 1% BSA and0.05% sodium azide and stored at 4° C. The following reaction equationsrepresent the synthesis of 2,7-dimethoxy-DMAE and conjugates.

Example 2 (a) Synthesis of 2-CME-7-methoxy-DMAE

The synthesis of 2-CME-7-methoxy-DMAE was accomplished from5-methoxyisatin and 4-bromophenol. N-Alkylation of the sodium salt of5-methoxyisatin with 4-benzyloxybromobenzene followed by rearrangementin hot alkali afforded the functionalized acridine carboxylic acid whichwas condensed with 4-nitro-2,6-dim ethyl phenol. The benzyl ether in theresulting acridine ester was first cleaved off and the free hydroxylgroup was alkylated with benzyl bromoacetate. Methylation of theacridine nitrogen with methyl triflate followed by conversion of thebenzyl ester to the free acid completed the synthesis. The acridiniumcompound was converted to the valproate conjugate via the NHS ester.

(b) Synthesis of 4-benzyloxybromobenzene

4-Bromophenol (2 g, 0.0116 mol) in acetone (40 mL) was treated withpotassium carbonate anhydrous (1.91 g, 1.2 equivalents) and benzylbromide (1.44 mL, 1.05 equivalents). The reaction was refluxed under anitrogen atmosphere. After 5-6 hours reflux, the reaction was cooled toroom temperature and diluted with an equal volume of ethyl acetate. Thissolution was washed with water, dried over anhydrous magnesium sulfateand evaporated to dryness. A white fluffy solid was obtained. Yield 2.36g (73%).

(c) Synthesis of N-[4′-benzyloxy)phenyl]-5-methoxyisatin

5-Methoxyisatin (1.5 g, 0.85 mmol) in anhydrous DMF (50 mL) was cooledin an ice bath under a nitrogen atmosphere and treated with sodiumhydride (0.25 g, 1.2 equivalents). The reaction was stirred in the icebath and after 15-20 minutes, 4-benzyloxybromobenzene (2.36 g, 0.85mmol) was added as a solution in anhydrous DMF (3 mL) along with copperiodide (3.23 g, 2 equivalents). The reaction was heated in an oil bathunder a nitrogen atmosphere at 130° C. for 24 hours. The reaction wasthen cooled to room temperature and filtered and the filtrate wasevaporated to dryness. The crude material was purified by flashchromatography on silica gel using 35% ethyl acetate in hexanes aseluent. The N-alkylated isatin derivative was isolated as anorange-brown solid. Yield=1 g (32%).

(d) Synthesis of 2-benzyloxy-7-methoxy acridine-9-carboxylic acid

The N-alkylated isatin from (b) above (1 g) was suspended in 10%potassium hydroxide (100 mL) and refluxed under a nitrogen atmosphere.After 4 hours, the reaction was cooled for 5-10 minutes and thenfiltered while still hot. A yellow precipitate separated in thefiltrate. The filtrate was diluted with ice and water and was thenacidified with a mixture of concentrated HCl and ice until a thickyellow precipitate separated out. This was allowed to stand for 15minutes and was then filtered using a medium porosity flitted glassfunnel. The product was subsequently rinsed with dry ether and then airdried. The resulting yellow powder was then transferred to a roundbottom flask, suspended in anhydrous toluene and evaporated to dryness.Yield=0.75 g (75%).

(e) Synthesis of 2-benzyloxy-7-methoxy-2′,6′-dimethyl-4′-nitrophenylacridine-9-carboxylate

2-Benzyloxy-7-methoxy acridine-9-carboxylic acid (0.38 g, 0.106 mol)from (c) above in anhydrous pyridine (30-40 mL) was treated withp-toluenesulfonyl chloride (0.404 g, 2 equivalents) at 0° C. undernitrogen. After ˜5 minutes, 2,6-dimethyl-4-nitrophenol (0.177 g, 1equivalent) was added and the reaction was warmed to room temperatureand stirred for 24 hours. The solvent was then removed under reducedpressure and the residue was dissolved in chloroform (50 mL). Thissolution was washed with 3% aqueous sodium bicarbonate and then 3%aqueous ammonium chlorides. The chloroform extract was then dried overanhydrous magnesium sulfate and evaporated to dryness. The crude productwas purified by preparative TLC on silica using 70% hexanes, 25%chloroform, 5% ethyl acetate. Yield=0.26 g (48%)

(f) Synthesis of 2-hydroxy-7-methoxy-2′,6′-dimethyl-4′-nitrophenylacridine-9-carboxylate

The 2-benzyloxy-7-methoxy acridine ester from (d) above (0.2 g) wasstirred in a mixture of 30% HBr/AcOH (10 mL) and dimethyl sulfide (5 mL)at room temperature for 4 hours. The product was then precipitated bythe addition of anhydrous ether followed by filtration. A bright yellowsolid was obtained. HPLC analysis using a 4.6 mm×30 cm C18 column and a30-minute gradient of 10%->70% MeCN/water (each containing 0.05% TFA) ata flow rate of 1 mL/min and UV detection at 260 nm, indicated producteluting at 25 minutes (starting material elutes at 34 minutes).Yield=0.24 g.

(g) Synthesis of2-[benzyloxycarbonyl]methyloxy-7-methoxy-2′,6′-dimethyl-4′-nitrophenylacridine-9-carboxylate

The compound from (e) above (0.163 g, 0.38 mmol) in anhydrous DMF (10mL) was treated with potassium carbonate anhydrous (65 mg, 1.2equivalents) followed by benzyl bromoacetate (66.2 uL, 1.1 equivalents).The reaction was heated in an oil bath at 65° C. under a nitrogenatmosphere. After one hour, HPLC analysis using the gradient describedabove indicated >85% conversion with the product eluting at 32 minutes.The reaction was then cooled to room temperature and the solvent wasremoved under reduced pressure. The residue was dissolved in chloroform(50 mL) and this solution was washed once with 3% aqueous ammoniumchloride and 3% aqueous sodium bicarbonate. The chloroform extract wasthen dried over anhydrous magnesium sulfate and evaporated to dryness.The crude product (0.3 g) was used as such for the next reaction.

(h) Synthesis of2-[benzyloxycarbonyl]methyloxy-7-methoxy-2′,6′-dimethyl-4′-nitrophenyl-10-methylacridinium-9-carboxylate

The crude acridine ester from (f) above (0.3 g, 0.53 mmol) was dissolvedin dichloromethane (˜5 mL) and treated with sodium bicarbonate (0.44 g,10 equivalents) and methyl trifluoromethane sulfonate (0.6 mL, 10equivalents). The reaction was stirred at room temperature for 16 hours.HPLC analysis using the gradient described above showed completeconversion with the product eluting at 25 minutes. The reaction was sofiltered through glass wool and the filtrate was evaporated to dryness.A bright yellow solid was recovered that was used directly for the nextreaction. MALDI-TOF MS 582.4 obs. (581.6 calc.).

(i) Synthesis of2-carboxymethylether-7-methoxy-2′,6′-dimethyl-4′-nitrophenyl-methylacridinium-9-carboxylate (2-CME-7-methoxy-DMAE)

The crude acridinium ester from (g) above was stirred in 30% HBr/AcOH(10 mL) at room temperature for 4-5 hours. Anhydrous ether was thenadded to precipitate the product which was collected by filtration. Thisprecipitate was rinsed several times with ether and then air dried. Areddish-yellow powder was obtained. HPLC analysis using the gradientdescribed earlier indicated clean conversion to product eluting at 20minutes. Yield=0.128 g; MALDI-TOF MS 492.2 obs. (491.5 calc.).

(j) Synthesis of 2-CME-7-methoxy-DMAE NHS ester

2-CME-7-methoxy-DMAE (10 mg, 16.6 umoles) in MeCN (2 mL) was treatedwith N-hydroxysuccinimide (2.9 mg, 1.5 equivalents) and DCC (17 mg, 5equivalents). The reaction was stirred at room temperature for 1-2 hoursby which time a fine precipitate of DCU had formed in the reaction. Thereaction was filtered through glass wool and the solution was evaporatedto dryness. MALDI-TOF MS 589.1 obs. (588.6 calc.).

(k) Synthesis of 2-CME-7-methoxy-DMAE-Valproate conjugate

An ice-cold solution of 6-amino-2-propylhexanoic acid (13 mg, 75 μmol)in sodium phosphate buffer (0.10 M, pH 7.4, 0.70 ml) was mixed with anice-chilled solution of 2-CME-7-methoxy-DMAE NHS ester (1.0 mg, 1.7μmol) in DMF (0.30 ml). This mixture was allowed to stir at roomtemperature overnight. The desired product was isolated from C-18reversed phase HPLC and lyophilized. MALDI-TOF MS 646.1 obsd. (645.7calcd). The following reaction equations represent the synthesis of2-CME-7-OMe-DMAE and valproate conjugate.

Example 3 pH Titrations of Acridinium Derivatives

This example details how the data in FIGS. 1 and 2 were obtained. FIGS.1 and 2 illustrate how the acridinium to pseudo-base transition iseffected as a function of pH by the placement of methoxy groups on theacridinium ester.

A typical protocol as illustrated for 2,7-dimethoxy-DMAE is as follows.A solution of 2,7-dimethoxy-DMAE (0.5 mg/mL in DMF) was prepared and 20uL of this solution was diluted with 100 uL of 25 mM phosphate buffer ofthe appropriate pH and 80 uL of DMF. The solution was allowed toincubate at room temperature for 1.5-2 hours and then the UV spectrumwas recorded using a Beckman model DU 7500 spectrophotometer. A 0.1 mLquartz mini-cell was used for the measurements. For each pH, the entireUV spectrum from 220-500 nm was recorded. The intensity of theabsorption band of the acridinium chromophore varied with variousacridinium derivatives. For 2,7-dimethoxy-DMAE this band was observed at410 nm whereas for acridinium derivatives, unsubstituted at theacridinium nucleus, this absorption band was observed typically at 363nm.

Example 4 Synthesis of 3-[9-({(3-carboxypropyl)[(4-methylphenyl)sulfonyl]amino}carbonyl)-10-acridiniumyl]-1-propanesulfonate-valproateconjugate (NSP-AS-valproate)

NSP-AS-NHS ester (5 mg, 7.32 umoles) and 6-amino-2-propylhexanoic acid(5 mg, 28.9 umoles) were combined in a 1:1 mixture of DMF and 0.2 Msodium bicarbonate (0.5 mL). The reaction was stirred at roomtemperature for 2 hours and then for 16 hours at 4° C. HPLC analysisusing a 4.6 mm×30 cm C18 column and a 40-minute gradient of 1.0%->60%MeCN/water (each containing 0.05% TFA) at a flow rate of 1 mL/min and UVdetection at 260 nm showed product eluting at 28 minutes. This waspurified by preparative HPLC and the HPLC fractions were lyophilized todryness. Yield=2.2 mg, MALDI-TOF MS 740 obs (739 calc.).

Example 5 (a) Synthesis of conjugate of 8-carboxypropyl theophylline and1,6-hexanediamine (theophylline-HD)

8-Carboxypropyltheophylline (40 mg, 150 mmoles) in DMF (1.5 mL) wastreated with N-hydroxysuccinimide (50 mg, 434 umoles) and DCC (50 mg,242 μmoles). The reaction was stirred at room temperature for one hour.To this solution, 1,6-hexanediamine (175 mg, 0.15 mmol) was added alongwith 0.2 M sodium bicarbonate (1.5 mL). The reaction was stirred at roomtemperature for 16 hours and was purified directly by preparative HPLCon a 20 mm×30 cm C18 column using a 40-minute gradient of 0%->40%MeCN/water (each containing 0.05% TFA) at a flow rate of 16 ml/min andUV detection at 260 ml. The product eluting at 21.5 minutes wascollected and the HPLC fractions were concentrated under reducedpressure and further dried under vacuum. Yield=50 mg (91%); MALDI-TOF MS365 obs. (365.5 calc.).

(b) Synthesis of NSP-AS-BD-theophylline conjugate

NSP-AS-NHS ester (1.8 mg, 2.82 umoles) and theophylline-HD (10 mg, 28.2μmoles) were combined in a DMF (0.3 mL). To this solution, 0.2 M sodiumbicarbonate (0.3 mL) was added. The reaction was stirred at roomtemperature for 3 hours. The product was purified on a 10 mm×30 cm C18column using a 40-minute gradient of 10%->60% MeCN/water (eachcontaining 0.05% TFA) at a flow rate of 2.3 mL/min and UV detection at260 nm. The HPLC fraction containing product eluted at 24 minutes whichwas collected and lyophilized to dryness. Yield=1.3 mg (50%); MALDI-TOFMS 932 obs. (933 calc.).

(c) Synthesis of NSP-AS-ED-carbamazepine conjugate

NSP-AS-NHS ester (2 mg, 2.93 umoles) and carbazepine-ED (10 mg, 35.8umoles) were combined in DMF (0.3 mL) and 0.2 M sodium bicarbonate (0.3mL) was added. The reaction was stirred at room temperature for 3 hours.The product was purified as described above for the theophyllineconjugate. The product eluting at 33 minutes was collected and the HPLCfractions were lyophilized to dryness. Yield=1 mg (40%); MALDI-TOF MS846 obs. (846 calc.).

The following reaction equations represent the synthesis of NSP-ASconjugates:

Example 6 (a) Homogeneous carbamazepine assay using2,7-dimethoxy-DMAE-HD-SA-ED-carbamazepine conjugate

Homogeneous assays were performed in a total volume of 200 uL of 10 mMphosphate containing 150 mM NaCl, 0.05% BSA and 0.01% sodium azide.Carbamazepine standards (0, 4.24, 8.47, 16.9, 33.9, 50.8, 93.2 uM) inhuman serum were diluted 20-fold into the assay buffer. The finalconcentrations of carbamazepine in the assay were 0, 0.212, 0.424,0.845, 1.695, 2.54, 4.66 uM. Assays were nm with either 0.02 uM or 0.002uM tracer with similar results. Binding reactions were initiated withthe addition of an anti-carbamazepine, mouse monoclonal antibody to afinal concentration of 0.1 uM. After 1 hour at room temperature,chemiluminescence was measured directly (25 uL) on a MLA1 (Magic LiteLuminimeter, Bayer Diagnostics, no filter) using a modified triggeringreagent comprising 3% hydrogen peroxide+0.5% cetyltrimethylammoniumchloride (CTAC) in 100 mM NaHCO₃. The zero carbamazepine standard andthe high carbamazepine standard were differentiated approximately 5-foldin signal in the dose response curve with reasonable assay precision(<6% CV).

A 4-parameter logistic (“4PL”) curve-fitting algorithm was employed tosimulate the observed dose response curve. The theoretical dose responsecurve was virtually superimposable on the observed dose response curve.The data for the homogeneous carbamazepine assay using an acridiniumester tracer appears in Table 3 and has been plotted graphically in FIG.3.

TABLE 3 Homogeneous Carbamazepine Assay Using Acridinium Ester TracerCarbamazepine, Mean Relative Light uM Units % CV 0 486107 3.56 4.24333000 3.27 8.47 181063 4.46 16.9 152220 3.14 33.9 125080 5.12 50.8109587 3.74 93.2 94320 3.93

(b) Homogeneous Carbamazepine Assay Using NSP-AS-HD-SA-ED-CarbamazepineConjugate

Homogeneous assays were performed in a total volume of 300 uL of 10 mMphosphate containing 150 mM NaCl, 0.05% BSA and 0.01% sodium azide.Carbamazepine standards (0, 4.24, 8.47, 16.9, 33.9, 50.8, 93.2 uM) inhuman serum were diluted 10-fold into the assay buffer. The finalconcentrations of carbamazepine in the assay were 0.424, 0.847, 1.69,3.39, 5.08, 9.32 uM. Assays were run with 0.2 nM tracer. Bindingreactions were initiated with the addition of an anti-carbamazepine,mouse monoclonal antibody to a final concentration of 70 nM. After 30minutes at room temperature, chemiluminescence was measured directly (25uL) on a MLA1 (Magic Lite Luminometer, Bayer Diagnostics, no filter)using a modified triggering reagent comprising 3% hydrogen peroxide+0.5%arquad in 100 mM NaHCO₃. The zero Carbamazepine standard and the highCarbamazepine standard were differentiated approximately 13-fold insignal in the dose response curve with reasonable assay precision (<8%CV). The data for the homogeneous carbamazepine assay using anacridinium sulfonamide tracer appears in Table 4 and has been plottedgraphically on FIG. 4.

TABLE 4 Homogeneous Carbamazepine Assay Using Acridinium SulfonamideTracer Carbamazepine, Mean Relative Light uM Units % CV 0 39775 0.594.24 201075 3.12 8.47 276635 7.76 16.9 400335 0.04 33.9 477355 6.00 50.8501350 3.04 93.2 521020 1.53

Example 7 (a) Homogeneous theophylline assay using2,7-dimethoxy-DMAE-HD-theophylline conjugate

In the homogeneous theophylline assay, reactions were conducted in atotal volume of 200 uL of buffer as described in Example 6. Theophyllinestandards in human serum (0, 6.94, 1.3.9, 27.7, 55.5, 111, 222 uM) werediluted 10-fold into the reaction to give final theophyllineconcentrations of 0, 0.694, 1.39, 2.77, 5.55, 11.1 and 22.2 uM.Different concentrations of the tracer and the antibody(anti-theophylline mouse monoclonal) were investigated to determine anoptimal concentration of each component which produced the maximumdeflection in signal between the zero and high theophylline standard. Amaximum enhancement in signal of 2-3-fold was observed by varying theconcentration of the tracer from 0.002 to 0.02 uM with an antibodyconcentration at 0.1 uM. By employing a tracer concentration of 0.002 uMand an antibody concentration of 0.1 uM, the observed dose-responsecurve was able to distinguish all the theophylline standards. Carvefitting utilizing the 4PL method gave excellent correlation between thetheoretical and the observed dose response curves from which theconcentration of theophylline in three ACS:180 ligand controls (BayerDiagnostics) were calculated. The calculated concentrations oftheophylline for the three controls were somewhat lower than theindicated concentrations. The data for the homogeneous theophyllineassay using an acridinium ester tracer appears in Table 5 and has beenplotted graphically in FIG. 5.

TABLE 5 Homogeneous Theophylline Assay Using Acridinium Ester TracerTheophylline, uM Mean Relative Light Units % CV 0 1582490 3.51 6.941485077 3.05 13.9 1371950 3.15 27.7 1268183 3.41 55.5 1084917 4.56 111973450 1.25 222 891833 1.10

(b) Homogeneous Theophylline Assay Using NSP-AS-HD-theophyllineConjugate

In the homogeneous theophylline assay, reactions were conducted in atotal volume of 300 uL of buffer as described in Example 6. Theophyllinestandards in human serum (0, 6.94, 13.9, 27.7, 55.5, 111, 222 uM) werediluted 10-fold into the reaction to give final theophyllineconcentrations of 0, 0.694, 1.39, 2.77, 5.55, 11.1 and 22.2 uM. Assayswere run with approximately 0.2 nM tracer. Binding reactions wereinitiated with the addition of an anti-theophylline, mouse monoclonalantibody to a final concentration of 70 nM. After a 30 minute incubationat room temperature chemiluminescence was measured directly (25 uL) onthe MLA1 (no filter) using the modified triggering reagent. The zero andhigh theophylline standards were differentiated approximately 5-fold insignal in the dose response curve with reasonable assay precision (<5%CV). The data for the homogeneous theophylline assay using an acridiniumsulfonamide tracer appears in fable 6 and has been plotted graphicallyin FIG. 6.

TABLE 6 Homogeneous Theophylline Assay Using Acridinium SulfonamideTracer Theophylline, uM Mean Relative Light Units % CV 0 61270 0.53 6.9477970 4.88 13.9 112920 2.94 27.7 139890 2.47 55.5 216630 1.78 111 2688352.45 222 312575 1.74

Example 8 (a) Homogeneous Valproate assay using2,7-dimethoxy-DMAE-valproate conjugate

The assay was conducted in 100 uL of buffer as described in Example 6.Valproate standards (0, 87.5, 175, 350, 700 and 1400 uM) in human serumwere diluted 10-fold into the assay buffer. A tracer concentration of0.2 uM was employed and the concentration of the anti-valproate, mousemonoclonal was 1 uM. Reactions were initiated by the addition of theantibody and after incubation at room temperature for 1 hour, thereactions were diluted 100-fold and measured as indicated earlier. Thezero and high valproate standard were differentiated by 10-fold insignal. Again assay precision was good and all the valproate standardscould clearly be distinguished from one another. The data for thehomogeneous valproate assay using an acridinium ester tracer appears inTable 7 and has been plotted graphically in FIG. 7.

TABLE 7 Homogeneous Valproate Assay Using Acridinium Ester TracerValproate, uM Mean Relative Light Units % CV 0 2226627 2.94 87.5 11791534.36 175 853790 3.97 350 645213 3.45 700 451783 1.35 1400 337943 5.24

(t) Homogeneous Valproate Assay Using NSP-AS-Valproate Conjugate

The assay was conducted in 300 uL of buffer as described in Example 6.Valproate standards (0, 87.5, 175, 350, 700 and 1400 uM) in human serumwere diluted 10-fold in to the assay buffer to give final valproateconcentrations of 0, 8.75, 17.5, 35, 70 and 140 uM. A tracerconcentration of approximately 0.2 nM was employed and the concentrationof the anti-valproate, mouse monoclonal was 70 nM. Reactions wereinitiated by the addition of the antibody and after a 30 minuteincubation at room temperature chemiluminescence was measured directly(25 uL) on the MLA1 (no filter) using the modified triggering reagent.The zero and high valproate standards were differentiated approximately3.4-fold in signal in the dose response curve with reasonable assayprecision (S % CV). The data for the homogeneous valproate assay usingan acridinium sulfonamide tracer appears in Table 8 and has been plottedgraphically in FIG. 8.

TABLE 8 Homogeneous Valproate Assay Using Acridinium Sulfonamide TracerValproate, uM Mean Relative Light Units % CV 0 182265 1.02 87.5 3708956.61 175 458255 0.55 350 518490 8.10 700 600600 3.41 1400 628925 4.26

Example 9 (a) Synthesis of Conjugate of Dabsyl Chloride and EthyleneDiamine (Dabsyl-ED)

Dabsyl chloride (25 mg, 0.077 mol, Aldrich) was added to ethylenediamine (36 ul, 7 equivalents) in dichloromethane (2 mL). After 10-15minutes, TLC (25% ethyl acetate in hexanes) indicated no startingmaterial. The reaction was concentrated under reduced pressure and theresidue was dissolved in MeCN. HPLC analysis using a 4.6 mm×30 cm C18column and a 30-minute gradient of 10%->100% MeCN/water (each containing0.05% TFA) showed a single product eluting at 13.5 minutes. The productwas purified by preparative HPLC and the HPLC fractions wereconcentrated to a small volume by rotary evaporation and thenlyophilized to dryness. Yield=35 mg (80%); MALDI-TOF MS 348.3 obs.(347.4 calc.).

(b) Synthesis of Dabsyl-ED-Glutarate-NHS Ester

Dabsyl-ED (15 mg, 26.1 umoles) in MeCN (3 mL) was treated withN,N-diisopropylethylamine (9.2 uL, 2 equivalents) followed by glutaricanhydride (9 mg, 3 equivalents). After 2 hours at room temperature, HPLCanalysis using a 4.6 mm×30 cm C18 column and a 30-minute gradient of10%->70% MeCN/water (each containing 0.05% TEA) at a flow rate of 1mL/min and UV detection at 260 nm showed complete conversion with theproduct eluting at 18 minutes, (starting material elutes at 16 minutes).To this solution N-hydroxysuccinimide (15 mg, 5 equivalents) was addedfollowed by DCC (27 mg, 5 equivalents). The reaction was stirred at roomtemperature. After one hour, HPLC analysis indicated ˜70% conversion.Additional DCC (13.5 mg, 2.5 equivalents) was added and the reaction wascontinued for another hour. The reaction was then filtered through glasswool and the product was purified by preparative HPLC. The HPLCfractions were lyophilized to dryness. Yield=14.8 mg (85%).

(c) Synthesis of Anti-Valproate-Dabsyl Conjugate

An anti-valproate monoclonal antibody (0.5 mg, 0.45 mL of 1.1 mg/mLstock in PBS pH 7.4) was diluted with 0.1 M sodium bicarbonate (0.45mL). This solution was treated with a 50 uL of a DMF solution ofdabsyl-ED-glutarate-NHS ester (2 mg/mL in DMF). The labeling reactionwas carried out for 2-3 hours at room temperature and then the conjugatewas isolated by gel-filtration chromatography on Sephadex G25 using 10mM phosphate pH 7 as eluent. The extent of dabsyl incorporation wasdetermined by MALDI-TOF MS which indicated ˜9 dabsyls per protein. Theconjugate was stored at 4° C.

(d) Homogeneous Valproate Assay Using Dabsyl-labeled Anti-valproateAntibody and 2-CME-7-methoxy-DMAE-valproate

Reactions were run in 0.2 mL of buffer as described in Example 6Valproate standards in sheep serum were employed at concentrations of 0,10, 25, 50, 100 and 150 ug/mL which correspond to 0, 68.5, 171, 342.5,685, 1027.5 uM. The tracer was employed at a concentration of 1.29 nM.The dabsyl-labeled antibody was added last at a final concentration of0.4 uM. The reactions were incubated at room temperature for one hourand were then read directly as described in Example 6. The data obtainedfor the homogeneous valproate assay using quenching was plottedgraphically in FIG. 9.

Example 10 Conjugates for CL Resonance Energy Transfer

(a) Biotin Conjugate of Naphthofluorescein

A solution of naphthofluorescein NHS ester (2.3 mg) in DMF (0.25 ml) wasmixed with a solution of biotin-jeffamine (conjugate of biotintriethylene glycol diamine, 15 mg) in DMF (0.25 ml) and carbonate buffer(0.10 M, pH 8.5, 0.30 ml). After this mixture was stirred at roomtemperature overnight, solvents were removed under vacuum, and theresidue was taken up in CH₃CN/water for HPLC purification (C-18 reversedphase chromatography). The isolated product was identified by MALDI-TOFmass spectrometry. (833.2 calc., 833.5 obs.)

(b) Biotin Conjugate of Acridinium Analog

Biotin-jeffamine (2.0 mg) and 2-CME-7-methoxy-DMAE NHS ester (0.5 mg)were dissolved in 0.50 ml of DMF, and the solution was stirred at roomtemperature over a period of three hours. After the solvents wereremoved under vacuum, the desired product was isolated from C-18reversed phase chromatography and con firmed by MALDI-TOF massspectrometry. (847.3 calc., 848.2 obs.)

(c) Preparation of anti-HCG(beta)-naphthofluorescein

0.30 ml of mouse monoclonal anti-HCG-beta (Bayer, #10244590, 10.2 mg/ml)in acetate buffer (10 mM, pH 5.5) was passed through a G-25 column (1cm×15 cm) for buffer exchange, eluted with phosphate buffer (0.10 M, pH8.0), and 2.0 ml of protein was collected with total of 2.8 mg ofantibody. One third of that collected sample was mixed withnaphthofluorescein-NHS in DMF (0.10 ml, 1.0 mg/1.5 ml, 20 eq. total).After two hours, the reaction mixture was washed with PBS (20 mM, pH7.4, 0.15 M NaCl, 0.05% NaN₃) through CentriCon 10 (0.66 ml, 0.60mg/ml). MALDI-TOF of the product indicated 4.5 dyes were incorporatedinto one antibody.

(d) Preparation of anti-HCG(whole)-2-CME-7-methoxy-DMAE

0.90 ml of anti-HCG (whole) at 3.5 mg/ml in 0.15 M phosphate-0.1% NaN₃,pH 7.4 was passed through a G-25 column (1 cm×15 cm) eluted withphosphate buffer (0.10 M, pH 8.0), and the collected fractions werepooled and concentrated through CentriCon 10 to a volume of 0.90 ml at2.3 mg/ml. One third of this sample was incubated with a sample of2-CME-7-methoxy-DMAE NHS ester in DMF (0.075 ml, 0.50 mg/0.70 ml, 20eq.) for two hours. Purification through G-25 column and concentrationthrough CentriCon 10 gave a product 0.21 mg (0.52 mg/ml) in PBS buffer(20 mM, pH 7.4, 0.15 M NaCl, 0.05% NaN₃). MALDI-TOF analysis showed 1.3labels were attached to the antibody.

Example 11 Titration of Avidin and Biotin-AE with Biotin-JfNpFL inHomogeneous CL Resonance Energy Transfer Reactions

Binding reactions (300 uL) were assembled in BSA-PBS (0.1% BSA-10 mMpotassium phosphate-0.15 M NaCl-0.05% sodium azide, pH 8) as shown inTable 9. Biotin-jeffamine-naphtofluorescein, abbreviated as“biotin-Jf-Np-FL, 100 uL” at varying levels and biotin-hexaethyleneglycol-2-CME-7-methoxy-DMAE, abbreviated as “biotin-AE, 100 uL” werepremixed and the competitive binding reactions (6-14) were initiated byaddition of 1 uM neutravidin (Pierce) or streptavidin (100 uL, Sigma)and mixing, followed by 30 minutes incubation at 37° C. Controlreactions (1-5) received the reagents shown and were brought to 300 uLwith BSA-PBS. The data appears in Table 9. The data for each reactionappears as a numbered row in Table 9, with the first row of datarepresenting reaction 1, the second row representing reaction 2 and soforth.

TABLE 9 CL Resonance Energy Transfer in a Biotin-AE:Avidin:Biotin-JfNpFLComplex 100 uL BSA- 0.025 uM Biot- PBS Biotin-AE JfNpFL 1 uM pH 8Neutravidin Streptavidin (uL) (uM) Avidin (uL) RLU/5s RLU/5s 1. 0 0 0300 2,230 2,230 2. 0 0 100 200 2,075 2,340 3. 100 0 0 200 22,585 22,5854. 0 1.000 0 200 2,185 2,265 5. 0 1.000 100 100 2,080 2,370 Signal/Signal/ Net RLU/5s BG Net RLU/5s BG 6. 100 0.000 100 81,710 1.00 8,1101.00 7. 100 0.005 100 97,085 1.18 11,275 1.39 8. 100 0.010 100 103,5501.27 14,430 1.78 9. 100 0.050 100 193,420 2.36 34,910 4.30 10. 100 0.250100 409,350 5.10 117,130 14.44 11. 100 1.000 100 456,335 5.59 367,07545.26 12. 100 1.500 100 848,860 10.39 357,275 44.05 13. 100 2.000 100851,365 10.42 436,045 53.77 14. 100 4.000 100 283,835 3.47 893,085110.12

CL was measured at 4° C. on a MLA1 with red sensitive PMT-tube(Hamamatsu R2228) and 2 filters (Corion cut-off filters, to read >650nm) using a single injection of 300 ul of 1% peroxide-0.5% CTAC-0.1 Msodium bicarbonate-0.05% sodium azide, ply 8.3. Net RLUs per 5 secondsshown are the counts with 2100 or 2300 RLU for background for avidinalone subtracted. BiotinNpFL alone also gave this low background and byitself was not enhanced by either avidin. See data for reactions 4 and 5in Table 9. Neutravidin alone enhanced biotin-AE CL 4-fold, whilestreptavidin alone decreased it ˜3-fold. See data for reactions 6 and 3in Table 9. The inclusion of biotin-JfNpFL gave a dose dependent furtherincrease in CL. See data reactions 7-14 vs reaction 6 in Table 9. Therange of biotin-JfNpFL discernable with biotin AE and streptavidin was˜2 to 3 orders of magnitude, with a lower detection limit of <0.005 uMin the 100 uL sample. See FIG. 10 which represents a plot of ‘CL RETfrom Biotin-AE to Biotin-jfNpFL in a Streptavidin Complex’.

As shown in FIG. 10, at optimum concentrations the RET signal withstreptavidin increased to a maximum of 110-fold and with neutravidin byup to 10-fold. These results suggest that a sandwich assay up to 3 logdynamic range may be possible if the RET binding complex can be reducedin size to that of the biotin:avidin complex.

The binding of biotin-AE alone to avidin caused a modulation in thechemiluminescence signal produced. Compare the data for reaction 3 withthat for reaction 6 in Table 9. The signal of 22,585 increased to 81,710with neutravidin and decreased to 8,110 with streptavidin.Interestingly, neutravidin enhanced chemiluminescence (480 nm), whilestreptavidin decreased it. Thus, site specific chemical modification ofantibodies may be useful to modulate the signal produced by bound CLlabel. Consequently, in this example the amount of chemiluminescence RET(>650 nm), when biotin-NpFL at varying levels in binding reactionscompeted with biotin-AE for avidins, also depended on the avidinspecies.

Example 12 Homogeneous Sequential Saturation Assay for Biotin via CLEnergy Transfer with Biotin-AE, Biotin-Jf-Np-FL and Neutravidin

Biotin (100 uL) at the indicated concentration and 100 uL of 1 uMneutravidin were mixed and incubated for 1 hr a 20° C. (Table 10). Thena solution of 25 nM biotin-AE and 250 nM biotin-Jf-Np-FL was added,mixed and followed by 30 min incubation at 37° C. Finally, readout wasperformed on the MLA1 as above. All background controls were low(reactions 1-4). Net RLUs are counts minus the 3285 RLU background forneutravidin alone. Initial binding of biotin reduced the amount oflabeled biotins bound in the next step, thereby reducing the RETperceptably above 2.5 uM biotin in a dose dependent manner.

Free biotin was allowed to compete with fixed levels of biotin-AE,biotin-NpFL and neutravidin. As shown in Table 10, biotin bound toneutravidin in place of some of the labeled conjugates reduced theotherwise enhanced energy transfer.

TABLE 10 Homogeneous Assay for Biotin via CL RET with Biotin-HEG-AE,Biotin-JfNpFL and Neutravidin Incubation: for 60 min. at 20° C. 30 min.at 37° C. 100 uL 1 uM BSA/PBS 25 nM Biotin-AE Biotin Neutravidin pH 8250 nM Biot-JfNpFL Rxn. (uM) (uL) (uL) (uL) CL SIGNAL RLU/5s 1 0 0 300 03,180 2 0 100 200 0 3,285 3 10 uM 0 200 0 3,480 4 10 uM 100 100 0 3,6755 0 0 200 100 22,265 Net RLU/5s (%) 6 0.0 100 100 411,995 100.0 7 0.1100 100 389,205 94.5 8 0.5 100 100 423,255 100.5 9 1.0 100 100 465,025112.9 10 2.5 100 100 384,720 93.4 11 5.0 100 100 30,040 7.3 12 7.5 100100 23,710 5.8 13 10.0 100 100 21,200 5.1 14 20.0 100 100 19,145 4.6

Example 13 (a) Homogeneous Immunoassay for HCG via CL Resonance EnergyTransfer

This example details a homogeneous assay for HCG where one antibody islabeled with an acridinium ester (2-CME-7-methoxy-DMAE) while the secondantibody is labeled with a fluorescent dye (naphthofluorescein)

Competitive binding reactions (300 uL) were assembled in BSA-PBS.Anti-HCG (wholo) (7504MR)-(2-CME-7-methoxy-DMAE)_(1.3) was premixed to10.4 ug/mL with anti-HCG beta (102445901)-(naphthofluorescein)_(4.5) to12.0 ug/mL final. Then HCG standards (200 uL) at the varyingconcentration indicated in Table 2 were added to 100 uL of the premixper tubes (quadruplicates, each standard), sealed, and followed by a mixand 1 hr incubation in a 37° C. water bath. Finally 2 tubes for eachstandard were measured in the MLA1 at 4° C. by read out with the singlereagent (pH 8.3) CL initiation as above. Five minutes later the othertwo tubes were read out by on the same instrument, using the single CLreagent readout where the signal reached maximum at 5000 mIU/ml, HCGwith a 2.7-fold increase over that at 0 HCG and then declined gradually.

1. A homogenous resonance energy transfer sandwich assay for thedetection or quantitation of a macromolecular analyte comprising thefollowing steps: (a) conjugation of an antibody specific for themacromolecular analyte with a chemiluminescent acridinium compound; (b)conjugation of a second antibody specific for the same macromolecularanalyte with an acceptor molecule; (c) complexation of the conjugateswith the macromolecular analyte in a sample to from a reaction mixture;(d) triggering the chemiluminescence of the binding complex reactionmixture by adding chemiluminescence triggering reagents in a pH range ofabout 6 to 10 wherein excited state energy is transferred from thechemiluminescent acridinium compound of the first conjugate to theacceptor molecule of the second conjugate, thereby causing lightemission from the acceptor molecule or light attenuation; (e) measuringthe amount of light with a luminometer; and (f) calculating theconcentration of the macromolecular analyte by comparing the amount oflight emitted from the reaction mixture with a standard dose responsecurve which relates the amount of light emitted to a known concentrationof the macromolecular analyte.
 2. The assay of claim 1, wherein saidacridinium compound is an acridinium ester.
 3. The assay of claim 1,wherein said acridinium compound is an acridinium sulfonamide.
 4. Theassay of claim 1, wherein the macromolecular analyte of interest isselected from the group consisting of proteins, nucleic acids,oligosaccharides, antibodies, antibody fragments, cells, viruses, andsynthetic polymers.
 5. The assay of claim 1, wherein said acceptormolecule is selected from the group consisting of fluorophores andquenchers.
 6. The assay of claim 4, wherein said acceptor molecule is afluorophore selected front the group consisting of fluorescein,naphthofluorescein, rhodamine, texas red or resorufin.
 7. The assay ofclaim 4, where said acceptor molecule is a quencher and said quencher isdabsyl.